Collagen-calcium chelate, its preparation method and application

Abstract

This invention discloses a collagen-calcium chelate, its preparation method, and its application. The collagen-calcium chelate is synthesized using collagen with a specific sequence and soluble calcium salt as raw materials via an aqueous synthesis method. It combines the water-locking, skin-improving, pigment-lightening, skin-elasticity-enhancing, and whitening effects of collagen with the skin-tissue regeneration, skin-integrity-maintaining, skin-barrier-strengthening, epidermal cell proliferation and differentiation-remodeling, skin extracellular matrix-remodeling, whitening, and soothing effects of calcium ions. This results in a collagen-calcium chelate with good stability and high biological efficacy, achieving the goals of whitening, moisturizing, repairing, and soothing.

Description

Technical Field

[0001] This invention relates to the field of synthetic biology, and more specifically, to a collagen-calcium chelate, its preparation method, and its application. Background Technology

[0002] Calcium is one of the most abundant mineral elements in the human body, totaling approximately 1300g, or 1.5-2.0% of body weight. Calcium ions promote platelet aggregation, accelerate thrombin synthesis, and promote early fibrin formation. Calcium ions also help stimulate collagen synthesis and angiogenesis. They can increase fibroblast metabolic activity, promote migration, and regulate MMP and cytokine release. Furthermore, calcium ions play a crucial role in many areas of skin function, including skin tissue regeneration, maintaining skin integrity, strengthening the skin barrier, promoting epidermal cell proliferation and differentiation, remodeling the skin's extracellular matrix, and whitening and soothing effects.

[0003] The main function of collagen is to lock in skin moisture, improve skin texture, lighten pigmentation, enhance skin elasticity, and whiten the skin. Many current studies focus only on the whitening and moisturizing effects of collagen alone or metal ions. This is not only cumbersome to use, but also prone to collagen degradation, resulting in unsatisfactory actual effects. However, chelating collagen with metals to create chelates not only greatly improves collagen stability but also allows the individual effects to be combined, achieving a synergistic effect greater than the sum of its parts. Furthermore, it is more convenient for users.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a collagen-calcium chelate, its preparation method, and its application. The collagen-calcium chelate obtained by the preparation method of this invention has the advantages of good stability and high biological efficacy, and can achieve the purposes of whitening, moisturizing, repairing, and soothing.

[0006] This invention is implemented as follows:

[0007] In a first aspect, the present invention provides a collagen-calcium chelate, wherein the collagen in the collagen-calcium chelate is derived from type I collagen, and its amino acid sequence is shown in SEQ ID NO: 1.

[0008] Secondly, the present invention provides a method for preparing the above-mentioned collagen-calcium chelate, which includes: using the above-mentioned collagen and soluble calcium salt as raw materials, and synthesizing the collagen-calcium chelate by an aqueous system synthesis method.

[0009] In some embodiments, the collagen is obtained by high-density fermentation of recombinant Escherichia coli that expresses the collagen.

[0010] In some embodiments, the soluble calcium salt is selected from at least one of calcium chloride and calcium acetate.

[0011] In some embodiments, the mass ratio of the collagen to the soluble salt is 10:1 to 3:1.

[0012] In some embodiments, the reaction conditions are as follows: stirring at 20–50°C for 0.5–4 h, and controlling the pH to be 4.5–6.5.

[0013] In some embodiments, the alkaline solution used to adjust the pH in the above reaction is selected from at least one of sodium hydroxide solution, potassium hydroxide solution, and barium hydroxide solution.

[0014] In some embodiments, the above preparation method further includes: after the reaction is completed, the reaction solution is subjected to dialysis or ultrafiltration, and then subjected to vacuum freeze-drying.

[0015] Thirdly, the present invention also provides the application of the above-mentioned collagen-calcium chelate in the field of cosmetics.

[0016] Fourthly, the present invention also provides the application of the above-mentioned collagen-calcium chelate in the food or pharmaceutical fields.

[0017] The present invention has the following beneficial effects:

[0018] This invention uses collagen with a specific sequence and soluble calcium salt as raw materials, and synthesizes collagen-calcium chelate using an aqueous system. It combines the water-locking, skin-improving, pigmentation-lightening, skin-elasticity-enhancing, and whitening effects of collagen with the skin-tissue regeneration, skin-integrity-maintaining, skin-barrier-strengthening, epidermal cell proliferation and differentiation-remodeling, skin extracellular matrix-remodeling, whitening, and soothing effects of calcium ions. This results in collagen-calcium chelate with good stability and high biological efficacy, achieving the goals of whitening, moisturizing, repairing, and soothing. Attached Figure Description

[0019] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1The image shows the infrared spectrum of collagen-calcium chelate. The horizontal axis represents wavenumber (cm⁻¹), and the vertical axis represents transmittance (%). Curve 1 is the collagen curve, and curve 2 is the collagen-calcium chelate curve. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0022] To obtain safer, more stable, and more effective cosmetics, this invention combines collagen with calcium and prepares a collagen-calcium chelate through an aqueous synthesis method, the specific method of which is as follows:

[0023] Collagen and soluble calcium salts are used as raw materials, which are mixed and reacted. After the reaction is completed, the reaction solution is dialyzed or ultrafiltered and then freeze-dried under vacuum.

[0024] The collagen used is derived from type I collagen, and its amino acid sequence is shown in SEQ ID NO: 1:

[0025] 。

[0026] During the experiment, it was found that compared with full-length type I collagen, the collagen fragments in this invention not only have the effects of full-length type I collagen, but also have the advantage of being more easily absorbed and utilized due to their smaller molecular weight. As a result, when prepared as collagen-calcium chelates, they have stronger whitening, moisturizing, repairing and soothing effects.

[0027] In this invention, the collagen used is produced by high-density fermentation of the genetically engineered bacterium BL21(DE3). Specifically, the construction method of the genetically engineered bacterium BL21(DE3) is as follows:

[0028] The gene of the collagen fragment with the amino acid sequence shown in SEQ ID NO: 1 was inserted into the expression vector to obtain the recombinant vector. The recombinant vector was then introduced into the starting strain BL21(DE3) to obtain the above-mentioned genetically engineered bacteria.

[0029] The method for producing collagen through high-density fermentation of genetically engineered bacteria is a conventional method in the field, and the present invention does not limit it, as long as the collagen can be obtained.

[0030] The soluble calcium salts used in the above preparation methods include, but are not limited to, calcium chloride and calcium acetate, and may also be other soluble calcium salts available in the art.

[0031] The collagen and soluble calcium salt were synthesized in an aqueous system to obtain a collagen-calcium chelate. In the reaction system, the mass ratio of collagen to soluble calcium salt was 10:1 to 3:1. Specifically, the mass ratio of collagen to soluble calcium salt could be 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1 or 3:1, or any other ratio between 10:1 and 3:1.

[0032] When the above-mentioned collagen and soluble calcium salt react, the reaction conditions are as follows: stirring at 20-50℃ for 0.5-4 hours, and controlling the pH to be 4.5-6.5.

[0033] Specifically, the reaction temperature can be 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, or 50℃, or any other value between 20℃ and 50℃. The reaction time can be 0.5h, 1h, 2h, 2.5h, 3h, 3.5h, or 4h, or any other value between 0.5h and 4h. The pH can be 4.5, 5, 5.5, 6, or 6.5, or any other value between 4.5 and 6.5.

[0034] In the above process, for pH adjustment, the alkaline solution includes, but is not limited to, sodium hydroxide solution, potassium hydroxide solution and barium hydroxide solution, and may also be other alkaline solutions available in the art.

[0035] After the reaction is complete, the reaction solution needs to be separated and purified to extract the collagen-calcium chelate. Optionally, the separation and purification process includes dialysis or ultrafiltration followed by vacuum freeze-drying. Specific methods and steps are not limited in this invention, as long as the collagen-calcium chelate can be separated from the reaction solution.

[0036] Through the above preparation method, this invention can obtain a collagen-calcium chelate, which has the advantages of good stability and high biological efficacy, and can achieve the purposes of whitening, moisturizing, repairing, and soothing. Therefore, this chelate can be applied in the fields of cosmetics or pharmaceuticals.

[0037] Specifically, products prepared from the collagen-calcium chelate of the present invention include cosmetics, food, or pharmaceuticals.

[0038] The aforementioned cosmetics include types such as face masks, serums, and facial cleansers. The collagen-calcium chelate of the present invention can be added alone to different types of cosmetics, or the collagen-calcium chelate of the present invention can be combined with other active ingredients and added to different types of cosmetics.

[0039] Alternatively, the above-mentioned collagen-calcium chelate can be added alone to different types of food, or the collagen-calcium chelate of the present invention can be combined with other active ingredients and added to different types of food.

[0040] Alternatively, the above-mentioned collagen-calcium chelate can be combined with pharmaceutically acceptable excipients to prepare a drug with whitening, moisturizing, repairing, and soothing effects. The above-mentioned collagen-calcium chelate can also be compounded with other active ingredients to prepare a drug with whitening, moisturizing, repairing, and soothing effects.

[0041] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0042] Example 1

[0043] This embodiment describes a method for preparing collagen-calcium chelates, and the specific steps are as follows:

[0044] (1) Weigh 100mg of collagen and dissolve it in 10mL of double-distilled water. Then add 1mL of calcium chloride solution with a volume concentration of 10mg / mL to the collagen solution. The reaction temperature is 20℃. The reaction is carried out for 1.5 hours under stirring. At the same time, add sodium hydroxide solution with a molar concentration of 0.5mol / L to control the pH value of the reaction solution to 4.0.

[0045] The amino acid sequence of collagen is shown in SEQ ID NO: 1.

[0046] (2) After the above reaction is completed, the reaction solution is dialyzed and then subjected to vacuum freeze-drying to obtain collagen-calcium chelate.

[0047] Example 2

[0048] This embodiment describes a method for preparing collagen-calcium chelates, and the specific steps are as follows:

[0049] (1) Weigh 100mg of collagen and dissolve it in 10mL of double-distilled water. Then add 1mL of calcium acetate solution with a volume concentration of 10mg / mL to the collagen solution. The reaction temperature is 20℃. The reaction is carried out for 1.5 hours under stirring. At the same time, add sodium hydroxide solution with a molar concentration of 0.5mol / L to control the pH value of the reaction solution to 4.0.

[0050] The amino acid sequence of collagen is shown in SEQ ID NO: 1.

[0051] (2) After the above reaction is completed, the reaction solution is dialyzed and then subjected to vacuum freeze-drying to obtain collagen-calcium chelate.

[0052] Example 3

[0053] This embodiment describes a method for preparing collagen-calcium chelates, and the specific steps are as follows:

[0054] (1) Weigh 30mg of collagen and dissolve it in 10mL of double-distilled water. Then add 1mL of calcium chloride solution with a volume concentration of 10mg / mL to the collagen solution. The reaction temperature is 20℃. The reaction is carried out for 1.5 hours under stirring. At the same time, add sodium hydroxide solution with a molar concentration of 0.5mol / L to control the pH value of the reaction solution to 4.0.

[0055] The amino acid sequence of collagen is shown in SEQ ID NO: 1.

[0056] (2) After the above reaction is completed, the reaction solution is dialyzed and then subjected to vacuum freeze-drying to obtain collagen-calcium chelate.

[0057] Comparative Example 1

[0058] This comparative example is collagen with the amino acid sequence shown in SEQ ID NO: 1.

[0059] Experimental Example 1

[0060] The collagen-calcium chelate obtained in Example 1 and the collagen in Comparative Example 1 were subjected to infrared spectroscopy analysis. The analysis results are as follows: Figure 1As shown, 1 represents collagen, and 2 represents collagen-calcium chelate.

[0061] The positions and intensities of the amide I and amide II bands in the collagen-calcium chelate were altered. This indicates that the absorption peaks of the functional groups in the protein molecule shift during chelate formation, suggesting an interaction between collagen and calcium that enhances collagen stability.

[0062] Experiment Example 2

[0063] This experiment compared the effects of collagen-calcium chelate obtained in Example 1, collagen from Comparative Example 1, and type I collagen (Sequence ID XP_063455318.1, full length 1507aa) on in vitro tyrosinase inhibition. The test method followed the requirements of the Guangdong Provincial Cosmetic Society Group Standard "T / GDCA 0062021 Test Method for Inhibition of Tyrosinase Activity by Cosmetic Raw Materials (In Vitro Method)". The in vitro tyrosinase inhibition rate was detected using collagen-calcium chelate, collagen, and the aforementioned type I collagen (Sequence ID XP_063455318.1, full length 1507aa) obtained from Comparative Example 1 and Comparative Example 2. The specific method is as follows:

[0064] A 96-well plate was configured with solvent background wells (Ta), solvent reaction wells (Tb), sample background wells (Tc), and sample reaction wells (Td). The Ta group was the solvent background group, containing neither L-tyrosine substrate solution nor sample solution; the Tb group was the solvent reaction group, containing L-tyrosine substrate solution but no sample solution; the Tc group was the sample background group, containing neither L-tyrosine substrate solution nor sample solution; and the Td group was the sample reaction group, containing both L-tyrosine substrate solution and sample solution. Each group was replicated three times.

[0065] Experimental steps:

[0066] As shown in Table 1, L-tyrosine solution, sample solution / solvent, and PBS buffer were added sequentially to each well. After thorough mixing, the mixture was incubated at 37°C for 10 min. Then, 20 μL of tyrosinase solution was added to each well, and the mixture was incubated at 37°C for 5 min ± 5 s. The wells were then immediately placed in a microplate reader for detection at 475 nm. The concentration of all samples was 0.2 mg / mL.

[0067] Note: The time from adding tyrosinase solution to measuring absorbance should be consistent for each well (5 min ± 5 s).

[0068] Table 1. Sample loading table for tyrosinase activity inhibition test

[0069]

[0070] Calculation of tyrosinase activity inhibition rate:

[0071] Y=(1-(Ad-Ac) / (Ab-Aa))×100%

[0072] In the formula: Y is the tyrosinase activity inhibition rate; Ad is the absorbance of the sample reaction well; Ac is the absorbance of the sample background well; Ab is the absorbance of the solvent reaction well; Aa is the absorbance of the solvent background well.

[0073] Table 2. Results of tyrosinase activity inhibition rates in Example 1, Comparative Example 1, and type I collagen.

[0074] experiment Tyrosinase activity inhibition rate (%) Example 1 (Collagen-Calcium Chelate) 43.18±0.34 Comparative Example 1 (Collagen) 34.48±0.27 Type I collagen 5.98±0.25

[0075] As shown in Table 2, compared with the same concentration of collagen and type I collagen (Sequence ID XP_063455318.1, full length 1507 aa) used in this invention, the collagen-calcium chelate can increase the inhibition rate of tyrosinase activity by 8.7% and 37.2%, respectively. This indicates that the collagen-calcium chelate has a better effect on inhibiting tyrosinase activity than the collagen used in this invention. Furthermore, it can be seen that the collagen and collagen-calcium chelate used in this invention have a superior effect on inhibiting tyrosinase activity compared to type I collagen (Sequence ID XP_063455318.1, full length 1507 aa).

[0076] Experimental Example 3

[0077] This experiment compares the effects of collagen-calcium chelate obtained in Example 1, collagen from Comparative Example 1, and type I collagen (Sequence ID XP_063455318.1, full length 1507aa) on the inhibition rate of melanin production in zebrafish.

[0078] Experimental Methods: Zebrafish were divided into four groups, including a normal control group and an experimental group. The normal control group received no treatment. The experimental groups were incubated with collagen-calcium chelate obtained in Example 1, collagen from Comparative Example 1, and type I collagen (Sequence ID XP_063455318.1, full length 1507aa) at a concentration of 0.1 mg / mL, respectively. After incubation for 48 hours, the zebrafish were photographed and the melanin signal intensity in the zebrafish heads was analyzed to evaluate the whitening effect.

[0079] Table 3. Results of melanin production inhibition rate of Example 1, Comparative Example 1, and type I collagen.

[0080]

[0081]

[0082] As shown in Table 3, compared with the same concentration of collagen (Sequence ID XP_063455318.1, full length 1507aa) and full-sequence type I collagen used in this invention, the collagen-calcium chelate can increase the inhibition rate of melanin production in zebrafish by 8.7% and 19.20%, respectively. This indicates that the collagen-calcium chelate is more effective than the collagen used in this invention in inhibiting melanin production in zebrafish. Furthermore, it demonstrates that the collagen-calcium chelate and collagen used in this invention have a superior effect on inhibiting melanin production in zebrafish compared to type I collagen (Sequence ID XP_063455318.1, full length 1507aa).

[0083] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A collagen-calcium chelate, characterized in that, The amino acid sequence of the collagen in the collagen-calcium chelate is shown in SEQ ID NO:

1.

2. The method for preparing collagen-calcium chelate as described in claim 1, characterized in that, include: The collagen-calcium chelate was prepared by using the collagen and soluble calcium salt as raw materials and an aqueous synthesis method.

3. The preparation method according to claim 2, characterized in that, The collagen is obtained through high-density fermentation of recombinant Escherichia coli that expresses the collagen.

4. The preparation method according to claim 2, characterized in that, The soluble calcium salt is selected from at least one of calcium chloride and calcium acetate.

5. The preparation method according to claim 2, characterized in that, The mass ratio of collagen to soluble calcium salt is 10:1 to 3:

1.

6. The preparation method according to claim 2, characterized in that, The collagen is mixed with soluble calcium salt and then reacted under the following conditions: stirring at 20-50°C for 0.5-4 hours, and the pH is controlled at 4.5-6.

5.

7. The preparation method according to claim 6, characterized in that, The alkaline solution used to adjust the pH in the reaction is selected from at least one of sodium hydroxide solution, potassium hydroxide solution, and barium hydroxide solution.

8. The preparation method according to claim 7, characterized in that, The preparation method further includes: after the reaction is completed, the reaction solution is subjected to dialysis or ultrafiltration, and then subjected to vacuum freeze-drying.

9. The application of the collagen-calcium chelate as described in claim 1 or the collagen-calcium chelate obtained by the preparation method according to any one of claims 2-8 in the cosmetics or food fields.

Images